The level of production of a protein in a host cell is determined by three major factors: the number of copies of its structural gene within the cell, the efficiency with which the structural gene copies are transcribed and the efficiency with which the resulting messenger RNA (“mRNA”) is translated. The transcription and translation efficiencies are, in turn, dependent on nucleotide sequences that are normally situated ahead of the desired structural genes or the translated sequence. These nucleotide sequences, also known as expression control sequences, define, inter alia, the locations at which RNA polymerase binds (the promoter sequence to initiate transcription; see also EMBO J. 5:2995–3000 (1986)) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation.
In most prokaryotes, the purine-rich ribosome binding site known as the Shine-Dalgarno (S-D) sequence assists with the binding and positioning of the 30S ribosome component relative to the start codon on the mRNA through interaction with a pyrimidine-rich region of the 16S ribosomal RNA. See, e.g., Shine & Dalgarno, Proc. Natl. Acad. Sci. USA 71:1342–46 (1976). The S-D sequence is located on the mRNA downstream from the start of transcription and upstream from the start of translation, typically from 4–14 nucleotides upstream of the start codon, and more typically from 8–10 nucleotides upstream of the start codon. Because of the role of the S-D sequence in translation, there is a direct relationship between the efficiency of translation and the efficiency (or strength) of the S-D sequence.
Not all S-D sequences have the same efficiency, however. Accordingly, prior attempts have been made to increase the efficiency of ribosomal binding, positioning, and translation by, inter alia, changing the distance between the S-D sequence and the start codon, changing the composition of the space between the S-D sequence and the start codon, modifying an existing S-D sequence, using a heterologous S-D sequence, and manipulating of the secondary structure of mRNA during the initiation of translation. Despite these changes, however, success in increasing of protein expression efficiency in prokaryotic systems has remained an elusive and unpredictable goal due to a variety of factors, including, inter alia, the host cells used, the expression control sequences (including the S-D sequence) used, and the characteristics of the gene and protein being expressed. See, e.g., Stenstrom, et al., Gene 273(2):259–265 (2001); Komarova, et al., Bioorg. Kbim. 27(4)282–290 (2001); Stenstrom, et al., Gene 263(1–2):273–284 (2001); and Mironova, et al., Microbiol. Res. 154(1):35–41 (1999). For example, efficient expression of soluble B. anthracis protective antigen (PA) has proved difficult in E. coli. See, e.g., Sharma, et al. Protein Expression and Purification 7:33–38 (1996) (indicating 0.5 mg/L at 70% purity); Chauhan, et al. Biochem. Biophys. Res. Commun.; 283(2):308–15 (2001) (indicating 125 mg/L); Gupta, et al. Protein Expr. Purif. 16(3):369–76 (1999) (indicating 2 mg/L).
Accordingly, there remains a demand in the art for compositions and methods for increasing the efficiency of ribosome binding and translation in prokaryotic systems, thereby resulting in increased efficiency of protein expression. This demand is especially strong for proteins that are difficult to express in existing systems, and for proteins that are desired in large quantity for pharmacological, therapeutic, or industrial use.